微小RNA-301a靶向Smad4促进前列腺癌细胞增殖的作用机制

The mechanism of microRNA-301a regulating the growth of human prostate cancer cells by targeting Smad4

目的探讨Smad4作为微小RNA(miRNA,miR)-301a调控的靶基因的证据,以及介导高糖促进前列腺癌细胞增殖的作用。方法采用TargetScan和PicTar数据库寻找miR-301a的分子靶点。采用转染miR-301a模拟物、实时定量聚合酶链反应及蛋白质印迹法(Western blot)法[25μg,10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)]检测miR-301a和Smad4的表达,双荧光素酶报道基因实验检测miR-301a对Smad4的调控,细胞计数试剂盒(CCK-8)法(10μl/孔CCK-8溶液,2 h)测定细胞增殖,以流式细胞术进行细胞周期分析。两组数据的均数检验采用t检验。结果生物信息学分析及荧光素酶报道实验均表明Smad4是miR-301a的靶点,上调miR-301a后,Smad4 mRNA及蛋白表达均明显降低。同时上调miR-301a及Smad4表达96 h后,前列腺癌细胞增殖能力升高160%(P<0.05),差异有统计学意义。miR-301a可抑制Smad4的表达,从而促进细胞从G1期向S期过渡。在PC-3细胞,转染miR-301a前的细胞周期比例为G0/G162.60%,S28.00%,G2/M 9.40%;转染后为G0/G149.97%,S 41.04%,G2/M 8.99%(转染前后G0和S期的比例差异有统计学意义,P<0.05);在DU-145细胞,转染miR-301a前G0/G165.16%,S 24.54%,G2/M 10.30%;转染后G0/G153.34%,S 36.67%,G2/M10.00%(转染前后G0和S期比例差异有统计学意义,P<0.05)。沉默Smad4的表达导致,细胞周期的G1期缩短,S期延长,与过表达miR-301a的结果相似;过表达Smad4可阻断miR-301a诱导的G1/S期转化。结论miR-301通过下调靶蛋白Smad4的表达来调控前列腺癌细胞增殖。Smad4在高糖相关的前列腺癌生长中发挥重要作用。

Objective To investigate the role of Smad4 as a target gene regulated by microRNA(miRNA,miR)-301a and the mediating effect of high glucose in promoting the proliferation of prostate cancer cells.Methods Real-time PCR was used to examine the expression of miR-301 in prostate cancer cells.Bioinformatics prediction by using software of TargetScan and PicTar and luciferase reporter gene assay was utilized for the identification of the target genes of miR-301.The expression of Smad4 was detected by real-time PCR and Western bloting[25μg,10%sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE)]after the miR-301 mimic was transfected 24 h later.After miR-301 mimics were transfected or co-transfected with Smad4 over-expression plasmid for 24 h,the proliferation and cell cycle of prostate cancer cells(PC-3 and DUl45)were examined by cell counting kit-8(CCK-8)and flow cytometry,respectively.Results Bioinformatics prediction and luciferase reporter gene assay demonstrated that Smad4 was the target of miR-301a.After up-regulating miR-301a,the expression of Smad4 mRNA and protein was significantly reduced.The cell proliferation ability increased by 160%(P<0.05)after up-regulating the expression of both miR-301a and Smad4 for 96 h.MiR-301a could inhibit the expression of Smad4,thereby promoting the cell transition from the G1 phase to the S phase.In PC-3 cells,before overexpression of miR-301a,the percentages of different phases were G0/G162.60%,S 28.00%,G2/M 9.40%;after overexpression,the percentages were G0/G149.97%,S 41.04%,G2/M 8.99%,the differences in the phases of G0 and S were significant(P<0.05).In DU-145cells,before overexpression of miR-301a,the percentages of different phases were G0/G165.16%,S 24.54%,G2/M 10.30%;after overexpression,the percentages were G0/G153.34%,S 36.67%,G2/M 10.00%,the differences in the phases of G0 and S were significant(P<0.05).In addition,knockdown of Smad4 shortened the G1 phase and prolonged the S phase,which was consistent with the results of miR-301a overexpression.Overexpression of Smad4 could block the effect of miR-301a inducing transformation of G1/S phase.Conclusion MiR-301 can regulate the proliferation of prostate cancer cells by targeting Smad4 gene.Smad4 plays an important role in the regulation of hyperglycemia associated prostate cancer.